polymeric micelles for the solubilization and delivery of stat3 inhibitor cucurbitacins in solid...

Polymeric micelles for the solubilization and delivery of STAT3inhibitor cucurbitacins in solid tumors

Ommoleila Molavia,b, Zengshuan Maa, Abdullah Mahmuda, Aws Alshamsana, JohnSamuela, Raymond Laic, Glen S. Kwond, and Afsaneh Lavasanifara,*

aFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G2N8

bBiotechnology Research Center, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

cDepartment of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada T6G1Z2

dSchool of Pharmacy, University of Wisconsin, Madison, WI 53705-2222, USA

AbstractPoly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) and newly developed poly(ethyleneoxide)-block-poly(α-benzyl carboxylate ε-caprolactone) (PEO-b-PBCL) micelles were evaluated forthe solubilization and delivery of cucurbitacin I and B, poorly water soluble inhibitors of signaltransducer and activator of transcription 3 (STAT3). Encapsulation of cucurbitacins in PEO-b-PCLand PEO-b-PBCL by co-solvent evaporation technique resulted in polymeric micelles <90 nm indiameter. The aqueous solubility of both derivatives increased from less than 0.05 mg/mL in theabsence of the copolymer to around 0.30–0.44 and 0.65–0.68 mg/mL in the presence of 5000–5000and 5000–24,000 PEO-b-PCL micelles, respectively. Maximum cucurbitacin solubilization wasachieved with PEO-b-PBCL micelles for both derivatives. PEO-b-PCL micelles having longer PCLblock were found to be more efficient in sustaining the rate of release for cucurbitacins. The anti-cancer and STAT3 inhibitory activity of polymeric micellar cucurbitacins were comparable with freedrugs in B16.F10 melanoma cell line in vitro. Intratumoral injection of 1 mg/kg/day cucurbitacin Iresulted in the regression of established B16.F10 mouse melanoma tumors in vivo. In comparison tofree cucurbitacin I, PEO-b-PBCL micellar cucurbitacin I was found to provide comparable anti-cancer effects against B16.F10 tumors and limit drug levels in animal serum while maintaining highdrug levels in tumor following intratumoral administration. The results indicate the potential ofpolymeric micelles as suitable vehicles for the delivery of cucurbitacin- I and B.

KeywordsPolymeric micelles; Cucurbitacin; Solubilization; Drug delivery; STAT3

1. IntroductionCucurbitacin- I and B (Fig. 1) are potent anti-cancer agents with selective inhibitory effect onsignal transducer and activator of transcription 3 (STAT3) pathway (Blaskovich et al.,2003;Jayaprakasam et al., 2003;Sun et al., 2005). Cucurbitacin I and B belong to a group ofnatural products called cucurbitacins, tetracyclic triterpinoid substances which have been

*Corresponding author. Tel.: +1 780 492 2742; fax: +1 780 492 1217. E-mail address:[email protected] (A.Lavasanifar).

NIH Public AccessAuthor ManuscriptInt J Pharm. Author manuscript; available in PMC 2009 April 1.

Published in final edited form as:Int J Pharm. 2008 January 22; 347(1-2): 118–127. doi:10.1016/j.ijpharm.2007.06.032.

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isolated from various plant families such as Cucurbitaceae. Cucurbitacins, possess a broadrange of potent biological activity derived largely from their cytotoxic properties (Chen et al.,2005). A number of compounds in this group have been investigated for their hepatoprotective,anti-inflammatory, anti-microbial and most importantly anti-cancer properties (Miro,1995;Duncan and Duncan, 1997;Jayaprakasam et al., 2003;Chen et al., 2005). The molecularmechanism of the various biological activities of cucurbitacins has not been fully investigated.Various functions of cucurbitacins have been related to their polarity and chemical structure(Dinan et al., 1997;Sun et al., 2005). For instance, it has been demonstrated that cytotoxiceffects of cucurbitacins increase linearly with their hydrophobicity (Bartalis and Halaweish,2005). Greige-Gerges et al. have also shown that the ability of cucurbtiacins to modify thebinding of bilirubin to albumin is related to their structure. In this study, cucurbitacin I wasfound not to affect the binding of bilirubin to albumin and considered to be less active inaffecting the interaction of albumin with small molecules in comparison to cucurbitacin E andD (Greige-Gerges et al., 2007). The anti-cancer activity of cucurbitacin I and B have beenshown in several cancer cell lines in vitro and there are some reports on their anti-tumor activityin vivo (Blaskovich et al., 2003;Jayaprakasam et al., 2003;Shi et al., 2006;Tannin-Spitz et al.,2007). Furthermore, cucurbitacin I and B have been shown to have selective signal transducerand activator of transcription 3 (STAT3) inhibitory activity in several cancer cell lines invitro (Blaskovich et al., 2003;Sun et al., 2005;Shi et al., 2006).

STAT3, a common oncogenic signaling pathway, is constitutively activated in many types ofcancers (Yu and Jove, 2004), including 82% of prostate cancers (Mora et al., 2002), 70% ofbreast cancers (Dolled-Filhart et al., 2003), more than 82% of the carcinomas of the head andneck (Nagpal et al., 2002), and 71% of nasopharyngeal carcinoma (Hsiao et al., 2003).Constitutive activation of STAT3 has been shown to play a major role in tumor cell growth,resistance to apoptosis and immune evasion by cancer. Accumulating evidence shows thatblocking aberrant activation of STAT3 in tumor results in the inhibition of cancer cell growth,induction of apoptosis and enhancement of anti-cancer immune responses (Yu and Jove,2004; Burdelya et al., 2005; Darnell, 2005; Kortylewski et al., 2005; Yu et al., 2007).Cucurbitacin I has been shown to modulate tumor-induced immunosuppression and enhanceanti-tumor activity of cancer immunotherapy in vivo (Nefedova et al., 2005). STAT3 inhibitoryand potent anti-proliferative activity of cucurbitacin I and B make them excellent and noveldrug candidates in cancer therapy. However, problems of poor water solubility and non-specifictoxicity have restricted their clinical benefit. Application of nanoscopic carriers such aspolymeric micelles for the delivery of cucurbitacins is expected to overcome both limitationsand enhance the therapeutic benefit of this important and emerging category of anti-cancerdrugs.

Polymeric micelles are nanoscopic carriers (20–100 nm in size) with a hydrophilic shell/hydrophobic core structure that have shown great promise in the solubilization and controlleddelivery of hydrophobic drugs (Aliabadi and Lavasanifar, 2006). Polyethylene oxide blockused as hydrophilic shell of micelles, masks the hydrophobic core from biological milieuleading to their prolonged circulation following intravenous (i.v.) administration. Longevityin blood circulation is followed by improved tumor accumulation through enhanced permeationand retention (EPR) effect leading to enhanced drug delivery with reduced toxicity (Nishiyamaet al., 2003; Hamaguchi et al., 2005). To date, only a limited number of polymeric micellarsystems have shown positive results in tumor targeted delivery of poorly soluble drugs aftersystemic administration (Aliabadi and Lavasanifar, 2006; Kwon and Forrest, 2006). The keyto success is to find the right drug–block copolymer combination that can withstand thedestabilizing effect of biological environment and provide a proper pattern of drug release inthe biological system.

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Poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) is a biocompatible copolymerwhich have been successfully used for the solubilization and controlled delivery of a numberof hydrophobic drugs (Allen et al., 1998, 2000; Kim et al., 1998; Aliabadi et al., 2005a). PEO-b-PCL micelles were also shown to cause a favorable shift in the pharmacokinetics andbiodistribution of cyclosporine A and hydroxylcampthotecin after i.v. administration (Aliabadiet al., 2005b; Shi et al., 2005). In this study, we have compared the potential of micelle-formingPEO-b-PCLs of different PCL molecular weights and the newly developed block copolymer,poly(ethylene oxide)-block-poly(α-benzyl carboxylate ε-caprolactone) (PEO-b-PBCL), asnanoscale drug delivery systems for the solubilization and controlled delivery of cucurbitacinI and B. The anti-cancer activity of polymeric micellar formulations as well as of cucurbitacinsin a mouse melanoma tumor model has also been evaluated and compared to the activity offree drug in vitro as we in vivo after intratumoral administration.

2. Materials and methods2.1. Materials

Cucurbitacin I (white powder with molecular weight of 514.7, soluble in acetone, DMSO,ethanol and methanol) was purchased from Calbiochem (San Diego, CA 92121, USA).Cucurbitacin B (white powder with molecular weight of 558, soluble in acetone and methanol)was obtained from PhytoMyco Research Corporation (Greenville, North Carolina, USA).Methoxy PEO (average molecular weight of 5000 g mol−1), diisopropyl amine (99%), benzylchloroformate (tech. 95%), sodium (in kerosin), butyl lithium (Bu-Li) in hexane (2.5 Msolution), palladium coated charcoal and thiazolyl blue tetrazolium bromide were purchasedfrom Sigma (St. Louis, MO, USA). Caprolactone was purchased from Lancaster Synthesis,UK. Stannous octoate was purchased from MP Biomedicals Inc., Germany. All other chemicalswere reagent grade.

2.2. Preparation and characterization of micellar formulations of cucurbitacin B and IPEO-b-PCL block copolymers having an identical PEO molecular weight of 5000 g mol−1 andPCL molecular weights of 5000 or 24,000 g mol−1 were synthesized and characterized asreported previously (Aliabadi et al., 2005a). PEO-b-PBCL block copolymer was alsosynthesized and characterized as described recently (Mahmud et al., 2006). A nomenclature,e.g., 5000–4700, 5000–5000 and 5000–24,000, in which the left number corresponds to thetheoretical molecular weight of the shell forming block and the right number corresponds tothe molecular weight of the core forming block, is used throughout the manuscript todistinguish between prepared block copolymers. Encapsulation of cucurbitacins in PEO-b-PCL (5000–5000 and 5000–24,000) and PEO-b-PBCL (5000–4700) micelles was achieved bya co-solvent evaporation method (Aliabadi et al., 2005a). Briefly 20 mg of copolymer and 2mg of either cucurbitacins B or I were dissolved in 0.5 mL acetone. This solution was addedto 2 mL doubly distilled water (ddH2O) in a drop-wise manner. After 4 h stirring at roomtemperature, the remaining acetone was removed by vacuum. The aqueous solution of themicellar formulation was then centrifuged at 3000 × g for 5 min to remove free cucurbitacinprecipitates. Polymeric micellar cucurbitacin formulations were used freshly in all in vitro andin vivo studies. Mean diameter and polydispersity of micelles were defined by light scattering(3000 HSA Zetasizer Malvern Zeta-Plus™ zeta potential analyzer, Malvern Instrument Ltd.,UK).

2.3. Determination of the cucurbitacin loaded levels by liquid chromatography–massspectrometry (LC–MS)

To determine the level of encapsulated cucurbitacin in PEO-b-PCL and PEO-b-PBCL micelles,aqueous solution of polymeric micelles were placed in centrifugal filter tubes (M. wt. cut-off= 100,000 g mol−1) and centrifuged at 3000 × g for 5 min to separate free and micelle-

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incorporated drug. Then 50 μL aliquot of the micellar solution (the top layer) was diluted in0.95 mL methanol to disrupt the micellar structure and release the incorporated drug. Dilutedsolution (0.1 mL) was added to 0.1 mL of 4-hydroxybenzophenone solution (0.01 mg/mLmethanol), which was used as internal standard (I.S.). This solution (10 μL) was injected toWaters Micromass ZQ 4000 LC–MS spectrometer. Quantitative analysis of cucurbitacin I byLC–MS was performed as described previously (Molavi et al., 2006). For the quantificationof cucurbitacin B by LC–MS, mass spectrometer was operated in negative ionization modewith selected ion recorder acquisition. Then the analytes were quantified with single ionrecording (SIR) at m/z 557 corresponding to [M – H] and m/z 539 corresponding to [M –H2O–H] for cucurbitacin B and at m/z 196.8 for I.S. For chromatographic separation a mobilephase consisting of a mixture of acetonitrile water containing 0.2% ammonium hydroxide(40:60) was employed for 3 min. This was followed by a non-linear gradient to a final ratio of60:40 (v/v) over 8 min at a constant flow rate of 0.2 mL/min. Calibration curves wereconstructed over the quantification range of 5–10,000 ng/mL for both cucurbitacin I and B.The ratios of cucurbitacin to I.S. peak areas were calculated and plotted versus cucurbitacinconcentration. Cucurbitacin loading and encapsulation efficiency were calculated by thefollowing equations:

2.4. In vitro release studyRelease study was preformed using dialysis method as reported previously (Forrest et al.,2006). As a control, free cucurbitacin I and B were dissolved in water at a concentration of 1mg/mL with the aid of methanol (2% v/v). The solubility of cucurbitacin I and B in watercontaining 2% methanol was verified using LC–MS. Aqueous solution of polymeric micellarcucurbitacin I and B were also prepared at a similar concentration. One milliliter of each samplewas placed in a Spectra/Pore dialysis bag (M.wt. cut-off = 12,000–14,000 g mol−1). Thedialysis bag was located in 100 mL ddH2O and whole system was placed in a rotating waterbath where temperature was kept at 37 °C during the experiment. In order to maintain “perfectsink” conditions, the bath was overflowed with fresh ddH2O so that the bath volume wasrefreshed every 1 h. Aliquots were taken at each time point from inside cassettes and drugconcentrations were measured by LC–MS. Three parallel measurements were performed ineach time point. Remaining drug in cassettes was determined and measurements were correctedfor drug removed in the previous samples.

2.5. Cell viability assayAnti-proliferative activity of free and PEO-b-PCL micellar cucurbitacin was assessed inB16.F10, a melanoma of C57/black origin (American Type Culture Collection, ATCC).B16.F10 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 IU/mL penicillin/streptomycin in 5% CO2 atmosphere. Cell viability wasmonitored using - 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT)assay (Mosmann, 1983). To evaluate cytotoxicity, B16.F10 cells were obtained fromexponentially growing 90–95% confluent cultures and seeded at a density of 2000 cells/wellin 96-well plates. After two days incubation the cells were washed twice in serum-free medium

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and treated with control vehicles (2% methanol and empty PEO-b-PCL micelles) or fourdifferent concentrations of either cucurbitacins I or B, dissolved in methanol or encapsulatedin 5000–24,000 PEO-b-PCL micelles (micellar cucurbitacin were diluted in cell culture mediaby serial dilution). After 24 or 48 h incubation at 37 °C the medium was removed and cellswere washed three times with the media followed by addition of 20 μL of 2 mg/mL solutionof MTT dye. The plates were returned to the incubator for a period of 4 h. The residual MTTsolutions were removed from wells then 0.2 mL of DMSO was added to each well and theplates were read at 570 nm using plate reader (PowerWave 340, Bio-Tek Instruments Inc.).Concentrations required for 50% inhibition in cell growth (IC50) were determined from log-linear dose–response curves for each agent.

2.6. Western blot analysisTo evaluate the effect of cucurbitacin I and B formulations on STAT3 phosphorylation,B16.F10 cells were plated into six-well tissue culture plates at 2 × 106 cells/well. Cells werethen incubated with cucurbitacin I at a concentration of 10 μM or cucurbitacin B atconcentration of 40 μM in either soluble form in 2% methanol or encapsulated in 5000–24,000PEO-b-PCL micelles for 24 h at 37 °C. Cells were then collected and washed twice with ice-cold PBS and then lysed in a buffer containing 30 mM Hepes (pH 7.5), 2 mM Na3VO4, 25mM NaF, 2 mM EGTA, 2% Nonidet P-40, 1:100 protease inhibitor mixture (Sigma–Aldrich),0.5 mM DTT and 6.4 mg/mL Sigma 104 Phosphatase Substrate (Sigma–Aldrich). Cell lysateswere centrifuged for 20 s at 16,000 × g. Total protein extract was determined by Micro BCAProtein Assay. Equal amounts of protein (20 μg) were loaded in 8% SDS-PAGE gel. SK-MEL-28 + IFN-γ cell lysate (Santa Cruz Biotech) was used as positive control. Proteins weretransferred into PVDF membrane and were probed with anti-p-STAT3 antibody (Santa CruzBiotech). To confirm equal loading, membranes were stripped and reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotech). Membraneswere developed using ECL plus detection kit (Amersham).

2.7. In vivo anti-tumor activityThe animal studies were carried out in accordance with procedures approved by the Universityof Alberta Health Sciences Animal Policy and Welfare Committee. A group of 16 C57Bl/6mice were injected subcutaneously with 106 B16.F10 melanoma cells at their left flank. Tumorswere allowed to grow until they became palpable. Animals were then randomly assigned tofour groups (4 mice per group). Control groups were kept untreated or received dailyintratumoral injection of empty PEO-b-PBCL micelles. Test groups were treated with 1 mg/kg cucurbitacin I in either soluble form (in 20% ethanolic solution) or encapsulated in PEO-b-PBCL micelles daily through intratumoral injection. Tumor size was measured duringtreatment three times a week by vernier caliper. Tumor volume (mm3) was calculated usingthe following equation:

Tumor-bearing animals were sacrificed 8–10 days after treatment and blood and tumor sampleswere collected. Cucurbitacin I was extracted from mice serum and tumor samples andquantified by LC–MS as described previously (Molavi et al., 2006).

2.8. Statistical analysisThe results are expressed as mean ± one S.D. for each group. The significance of differencesamong groups was analyzed either by one-way or two-way analysis of variance (ANOVA)

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followed by the Student–Newman–Keuls post hoc test for multiple comparisons. Beforeexecuting the ANOVA, data were tested for normality and equal variance. If any of those testsfailed, data were compared using a Kruskal–Wallis one-way ANOVA on ranks. A P value of≤0.05 was set for the significance of difference among groups. The statistical analysis wasperformed with SigmaStat software (Jandel Scientific, San Rafael, CA).

3. Results3.1. Encapsulation of cucurbitacin B and I in polymeric micelles

Table 1 summarizes the characteristics of 5000–5000 and 5000–24,000 PEO-b-PCL and 5000–4700 PEO-b-PBCL micelles loaded with cucurbitacin B and I. Polymeric micelles were ableto increase the water solubility of cucurbitacins. The aqueous solubility of both derivativesincreased from less than 0.05 mg/mL in the absence of the copolymer to around 0.30–0.44 and0.65–0.68 mg/mL in the presence of 5000–5000 and 5000–24,000 PEO-b-PCL micelles,respectively. Water solubility of cucurbitacin I in the presence of 5000–5000 PEO-b-PCL wasfound to be higher than that of B derivative (0.44 mg/mL versus 0.30 mg/mL) (P < 0.05,ANOVA). Between the two PEO-b-PCL micelles, those having longer PCL blocks were foundto be more efficient in encapsulating both cucurbitacin derivatives. Maximum cucurbitacinsolubilization was achieved with PEO-b-PBCL micelles for both derivatives (Table 1) despitea lower molecular weight of the PBCL block (4700 g mol−1). The difference in thesolubilization of cucurbitacins by 5000–4700 PEO-b-PBCL in comparison to 5000–24,000PEO-b-PCL was more evident for the more hydrophobic derivative; i.e, cucurbitacin B. Theaqueous solubility of cucurbitacins B and I was found to be 0.92 and 0.74 mg/mL in thepresence of PEO-b-PBCL micelles corresponding to an encapsulation efficiency of 92.9 and74.1%, respectively.

3.2. In vitro release of cucurbitacin B and I from polymeric micellar formulationsThe release profile of cucurbitacin B and I from different vehicles evaluated by dialysis methodare shown in Fig. 2A and B, respectively. The transfer of solubilized cucurbitacins frommethanolic solution through the dialysis bag was found to be relatively rapid (>60 and 90%transfer in 1 and 4 h, respectively, for both derivatives). In general polymeric micelles wereable to reduce the rate of drug transfer from the dialysis membrane to outside medium in thein vitro release experiment. For cucurbitacin B, the 5000–5000 PEO-b-PCL micelles showeda burst release (42% release) within 1 h followed by an accumulative 64% of encapsulateddrug release within 8 h. An increase in the molecular weight of PCL block from 5000 to 24,000g mol−1 significantly reduced the burst release of cucurbitacin B in the first hour of experimentfrom 42 to 23% and resulted in a significant decrease in accumulative drug release (P < 0.0001,ANOVA). This system released 40% of its drug content within 8 h (Fig. 2A). The releaseprofile of cucurbitacin B from PEO-b-PBCL micelles was similar to that for 5000–5000 PEO-b-PCL micelles, i.e., a burst release (around 45%) within 1 h followed by an accumulative 64%of encapsulated drug release within 8 h. In the case of cucurbitacin I, 5000–5000 PEO-b-PCLmicelles, the pattern of drug transfer showed minimal differences with the transfer of free drug.In this case, a burst release of 55% in the first hour followed by 90% drug release within 8 hwas observed (Fig. 2B). PEO-b-PCL micelles having 24,000 g mol−1 of PCL significantlyreduced the burst release of cucurbitacin I as well as accumulative drug release percent in 8 h(P < 0.0001, ANOVA). This system showed a burst release of 30% within 1 h followed by anaccumulative release of 80% for incorporated cucurbitacin I in 8 h. Micelles of PEO-b-PBCLshowed a similar release profile for cucurbitacin I to that of 5000–5000 PEO-b-PCL micellesin initial time points (50% drug release at 1 h), but a decreased rate of cucurbitacin I release atlater times (80% drug release at 8 h) (Fig. 2B).

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3.3. Anti-proliferative activity of free and polymeric micellar cucurbitacin B and I againstB16.F10 cell line in vitro

Fig. 3A and B depicts the anti-proliferative activity of methanolic solution or polymericmicellar formulation of cucurbitacin I and B against B16.F10 cells after 24 and 48 h incubation.Treatment of the murine B16.F10 melanoma cells with increasing concentrations ofcucurbitacin I or B as free or encapsulated in 5000–24,000 PEO-b-PCL micelles resulted in asignificant loss of cell viability assessed by MTT assay. Cucurbitacin I exerted more potentanti-proliferative activity than cucurbitacin B against the B16.F10 melanoma cells. Treatmentof B16.F10 cells with cucurbitacin I in methanolic solution for 24 h at concentrations of 1, 5,10 and 20 μM reduced the viability of treated cells to 81, 51, 43 and 22% of untreated cells,respectively (Fig. 3B), whereas cucurbitacin B incubation with B16.F10 melanoma cells forthe same incubation period at concentrations of 1 and 5 μM did not result in any significantloss of cell viability (data not shown) and the presence of B derivative at concentrations of 10and 20 μM left 64 and 47% of B16.F10 cells viable, respectively (Fig. 3A). As it has beenshown in Table 2 the IC50 values for cucurbitacin I against the B16.F10 melanoma cells after24 and 48 h incubation were significantly lower than those for cucurbitacin B at the sameincubation time (P < 0.0001, ANOVA). The IC50 values of free cucurbitacins B and I after 24h incubation were 18.9 and 4.7 μM. After 48 h incubation, the IC50 was calculated at 16.1 and4.6 μM for the B and I derivatives, respectively.

Similar difference between the activity of encapsulated cucurbitacin I and B was observed.Treatment of B16.F10 cell with polymeric micellar cucurbitacin B at concentrations of 10, 20,30 and 50 μM for 24 h reduced the cell viability to 83, 63, 61 and 58%, which were significantlyhigher than the viability of the cells treated with free drug at identical concentrations (P <0.0001, ANOVA). However, when the incubation time was increased to 48 h, the viability ofB16.F10 cells treated with polymeric micellar cucurbitacin B was not significantly differentfrom that of free drug (Fig. 3A) (P > 0.05, ANOVA). When B16.F10 cells were treated withpolymeric micelles of cucurbitacin I, at concentrations of 1, 5, 10 and 20 μM, the viability ofcells was 91, 60, 56 and 32% of untreated cells after 24 h incubation, respectively. As Fig. 3Bindicates, the viability of B16.F10 cells treated with free cucuribitacin I at the sameconcentrations for the same period of time were significantly lower than what was observedwith polymeric micellar cucurbitacin I (P < 0.0001, ANOVA). Treatment of B16.F10 cell withpolymeric micellar cucurbitacin I at the same concentrations for 48 h left 84, 53, 40 and 24%of the cells viable, which were not significantly different from what was observed with freecucurbitacin I (P > 0.05, ANOVA). As illustrated in Table 2, IC50 values for free form of bothderivatives are significantly different from those for their polymeric micellar formulation after24 h incubation (P < 0.05, ANOVA). However, there is no significant differences in IC50 valuesbetween free and encapsulated forms of both derivatives after 48 h incubation (P > 0.05,ANOVA). PEO-b-PCL and PEO-b-PBCL block copolymers did not show any cytoxicityagainst B16.F10 cells when incubated at concentrations as high as 100 μg/mL or below (datanot shown).

3.4. STAT3 inhibitory activity of free and polymeric micellar formulations of cucurbitacin Band I in B16.F10 cell line in vitro

Cucurbitacin I and B are selective inhibitors of Junes kinase (JAK)/STAT3 pathway and blockSTAT3 activation through reduction in the phosphorylated STAT3 (p-STAT3) level in tumorcells. Measuring the level of p-STAT3 in B16.F10 cells using Western blot analysis providedmeans to evaluate the anit-STAT3 activity of cucurbitacin formulations in this study. As it isshown in Fig. 4A, treatment of B16.F10 cells with free or in 5000–24,000 PEO-b-PCL micellarcucurbitacin B at concentration of 40 μM for 24 h resulted in the suppression of p-STAT3level. Free and 5000–24,000 PEO-b-PCL micellar cucurbitacin I were found to reduce the levelof p-STAT3 at a concentration of 10 μM (Fig. 4B).

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3.5. In vivo anti-tumor activity of cucurbitacin I and its micellar formulation in B16.F10melanoma mouse model after intratumoral injection

Based on the result of in vitro anti-cancer and STAT3 inhibitory activity, between twocucurbitacins, derivative I was more potent against cell growth as well as reduction of p-STAT3level in B16.F10 cell line. Therefore, this derivative was chosen for in vivo efficacy study ina B16 melanoma tumor model. Among all the micellar formulations evaluated for theencapsulation of cucurbitacin I, PEO-b-PBCL micelles was the most efficient one in thesolubilization of this compound and the level of drug release from this polymeric micellarsystem was also relatively slow and, except for the initial burst effect, was comparable to thatof 5000–24,000 PEO-b-PCL micelles. Thus, this block copolymer was selected to form themicellar structure of choice for cucurbitacin I delivery during in vivo studies. As it has beenshown in Fig. 5, B16 tumors from the mice in control groups, either untreated or treated withempty micelles, grew to about 3200 mm3 within 14 days after tumor implantation, whereasthe tumor growth was efficiently inhibited in both test groups that received 1 mg/kg/day ofintratumoral cucurbitacin I (~25 μg/mouse) as free or encapsulated in PEO-b-PBCL micelles.In fact, at the end of treatment period, in groups that received free or polymeric micellarcucurbitacin, 2 and 1 out of 4 mice were tumor free, respectively (Fig. 5C). Average tumorsize in mice treated with free and encapsulated cucurbitacin I was below 150 mm3 at day 17after tumor cell injection. The average weight of tumors in control animals that were untreatedor received empty PEO-b-PBCL micelles was five times higher than that in test groups treatedwith free or polymeric micellar cucurbitacin I (Fig. 5B and C). Quantification of cucurbitacinI in the serum samples obtained from mice (n = 4) in test groups 24 h after the last treatmentrevealed detectable level of cucurbitacin I in the serum of mice treated with free drug (58 ± 24ng/mL), but cucurbitacin I was below detectable level in the serum of animals receiving themicellar formulation of this drug (<10 ng/mL). Tumor samples from mice in the test groups(animals that received free and encapsulated drug and had visible tumor at the end of treatmentperiod) was harvested and analyzed for drug concentration 24 h after the last treatment.Cucurbitacin I was present at a concentration of 0.9 μg/g tumor tissue isolated from animalsreceiving free drug. The level of drug in tumor sample isolated from animals treated with PEO-b-PBCL micellar cucurbitacin I was 2.0 μg/g tumors.

4. DiscussionCucurbitacins I and B are of great interest due to their selective STAT3 inhibitory activity andstrong anti-proliferative function against a number of human carcinoma cell lines (Blaskovichet al., 2003; Jayaprakasam et al., 2003; Sun et al., 2005). The IC50 values of cucurbitacinsagainst several cancer cells are comparable with doxorubicin, a widely used anti-cancer drug(Jayaprakasam et al., 2003). Moreover, selective STAT3 inhibitory activity of cucurbitacinsmakes them excellent drug candidates for delivery to tumor microenvironment to overcometumor-induced immunosuppression, which may eventually lead to potent anti-tumor immuneresponses through inhibition of STAT3 (Yu et al., 2007). The main limitations to the clinicalapplication of cucurbitacins are their poor water solubility and non-specific toxicity.Development of a polymeric micellar carrier that can solubilize cucurbitacins effectively andcontrol their rate of release was purposed to overcome both limitations. For this purposemicelle-forming PEO-b-poly(ester)s having different poly(ester) blocks (PCL molecularweights of 5000 or 24,000 g mol−1 and PBCL molecular weight of 4700) were utilized (Fig.1).

Between two PEO-b-PCLs, those with longer hydrophobic block showed better efficiency forthe solubilization of both cucurbitacins (Table 1). This is predictable since longer PCL blockprovide more hydrophobic core leading to better interaction with hydrophobic molecules inthe micellar core. Besides, owing to a lower critical micellar concentration (CMC), block

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copolymers having more hydrophobic core structures may form of higher number of micellesat a certain concentration (assuming similar aggregation numbers) and load higher drug contentin total as a result.

PEO-b-PBCL micelles were found to be the most efficient vehicle for the solubilization of bothcucurbitacins despite higher CMC of 5000–4700 PEO-b-PBCL compared to 5000–24,000PEO-b-PCL (Mahmud et al., 2006; Aliabadi et al., 2005a). The presence of aromatic ring onPCL block increases the hydrophobicity and compatibility of core forming block with thehydrophobic structure of cucurbitacin derivatives. More effective solubilization of cucurbitacinB (the more hydrophobic derivative) in PEO-b-PBCL micelles provided further evidence forthis explanation.

Consistent with findings on the solubilization of cucurbitacin derivatives by polymericmicelles, PEO-b-PCL based polymeric micelles were found to be more efficient in controllingthe rate of release for the B derivative. In general, PEO-b-PCL micelles having longer PCLblocks were found to be more efficient in sustaining the rate of release for both drugs, invitro (Fig. 2). The rate of cucurbitacin B release from PEO-b-PBCL micelles was comparableto that of 5000–5000 PEO-b-PCL. For cucurbitacin I, the rate of drug release from PEO-b-PBCL was similar to 5000–5000 PEO-b-PCL micelles at the initial time point (<1 h) but itbecame slow and similar to that of 5000–24,000 PEO-b-PCL micelles afterwards.

In the next step, we assessed the in vitro anti-cancer and STAT3 inhibitory activity of free and5000–24,000 PEO-b-PCL micellar cucurbitacins I and B in B16.F10 melanoma cells in whichSTAT3 has been shown to be constitutively activated (Niu et al., 1999). Our results indicateda more potent anti-proliferative activity characterized by a significantly lower IC50 value forcucurbitacin I in comparison to cucurbitacin B (Table 2). Treatment of B16.F10 cells withpolymeric micellar cucurbitacin I resulted in a comparable loss of cell viability to that of freedrug at equal concentrations when cells were treated for 48 h. However, in shorter incubationtime (24 h), free cucurbitacin I was found to have better anti-proliferative activity thanencapsulated drug (Table 2). Identical findings were observed for cucurbitacin B (Table 2).The higher IC50 values of polymeric micellar cucurbitacins in shorter incubation times (24 h)may reflect slow release of incorporated drug from the micellar formulation (Fig. 2).Investigation of anit-STAT3 activity of cucurbitacin I and B formulations in B16.F10 cell line,revealed the activity of encapsulated cucurbitacins in reducing the level of p-STAT3 (Fig. 4).Taken together, the results of anti-cancer and STAT3 inhibitory activity show the ability ofmicellar formulations in the delivery of functional cucurbitacins to tumor cells.

The in vivo anti-cancer activity of more potent derivate, cucurbitacin I, and its PEO-b-PBCLmicellar formulation was also assessed in a mouse melanoma tumor model followingintratumoral injection of free and encapsulated drug. The selection of PEO-b-PBCLformulation for in vivo studies was made based on the higher efficiency of PEO-b-PBCLmicelles in the solubilization of cucurbitacin I. Our results indicated that intratumoral injectionof 1 mg/kg/day cucurbitacin I, as free or encapsulated in PEO-b-PBCL micelles, efficientlyinhibits the growth of B16.F10 tumor, in vivo (Fig. 5). This is consistent with a pervious studythat show intraperitoneal injection of 1 mg/kg/day of cucurbitacin I inhibits tumor growth ina mouse melanoma tumor model (Blaskovich et al., 2003). The tumor growth inhibitory effectof STAT-3 inhibitor cucurbitacin may be a result of direct anti-cancer activity and/or inductionof anti-tumor immune response by the drug. Further studies are required to define the possibleextent of contribution from both mechanisms.

Quantitative analysis of cucurbitacin I in the serum samples of the animals treated withcucurbitacin I revealed detectable drug level in the serum of the mice treated with free drug,but drug level was below detectable concentration in the serum of mice treated with

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cucurbitacin I micelles. On the other hand, the level of cucurbitacin I in tumors isolated fromthe mice treated with cucurbitacin I micelles was found to be higher than animals that receivedfree drug. This points to the retention of micellar cucurbitacin I within the tumor tissue and alower dissemination of drug encapsulated in micelles to the organs following its intratumoraladministration which may lead to a lower systemic toxicity for the polymeric micellarformulation of cucurbitacin I. Limited in vivo studies from our laboratory implied a potentialfor PEO-b-PCL micelles in causing a favorable change in the pharmacokinetic profile ofcucurbitacin I and a reduction in its in vivo toxicity after systemic administration (Molavi etal., 2006). These findings are consistent with previous reports on the in vitro release andpharmacokinetics of PEO-b-PCL micellar formulation of cyclosporine A andhydroxycompthotecin (Aliabadi et al., 2005b; Shi et al., 2005). It is; however, not clear whetherthis degree of change in drug release and normal pharmacokinetics of cucurbitacin I by itspolymeric micellar formulation will be sufficient to increase drug accumulation in solid tumorand reduce cucurbitacin toxicity after systemic administration. Further studies are required toassess the potential of polymeric micellar formulations of cucurbitacin I in enhancing itstherapeutic index after intravenous administration.

5. ConclusionOur results reveal a potential for PEO-b-PCL based micelles, especially PEO-b-PBCL andPEO-b-PCL having longer PCL blocks, as suitable vehicles for the solubilization andcontrolled delivery of cucurbitacin I and B. PEO-b-PCL micelles were shown superiority incontrolling the rate of drug release for the more hydrophobic derivative, i.e., cucurbitacin B,but the I derivative was found to be more potent in suppression of p-STAT3 level and inhibitionof cell proliferation in a STAT3 over expressing murine cancer cell line. In comparison to freedrug, PEO-b-PBCL micellar cucurbitacin I was found to provide comparable anti-cancereffects against B16.F10 tumors in mice, limit drug levels in animal serum while retainingenhanced drug levels in tumor tissue after intratumoral administration.

AcknowledgementsThis work was supported by research grants from Canadian Institute of Health Research (CIHR, grant MOP 42407)and the National Science and Engineering Research Council (NSERC, grants G121210926 and G121220086). O.M.has been supported by Rx and D HRF/CIHR graduate student research scholarship and a scholarship from IranianMinistry of Health and Medical Education.

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Fig. 1.Chemical structure of (A) cucurbitacin B; (B) cucurbitacin I; (C) PEO-b-PCL; and (D) PEO-b-PBCL.

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Fig. 2.The effect of solubilizing vehicle on the in vitro rate of (A) cucurbitacin B and (B) cucurbitacinI release. Data shown are representative of three independent experiments and the values foreach time point are mean of triplicates ± S.D.

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Fig. 3.Anti-cancer activity of free and polymeric micellar (A) cucurbitacin B; (B) cucurbitacin Iformulations against B16 melanoma cell line after 24 and 48 h incubation, in vitro. B16 cellswere treated with five different concentrations of either cucurbitacin I or B in free or polymericmicellar form. After 24 or 48 h incubation, cell viability was estimated by MTT assay andexpressed as percentage of untreated controls. The data represent the mean ± S.D. of threeindependent experiments.

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Fig. 4.Inhibition of p-STAT3 in B16.F10 melanoma cells by soluble (Sol) and polymeric micellar(Mic) (A) cucurbitacin B at a concentration of 40 μM and (B) cucurbitacin I at a concentrationof 10 μM. Methanol-treated group (Methanol) or empty-micelle treated group (Mic.0) werecontrols. SK-MEL-28 + IFN-γ cell lysate was used as positive control. p-STAT3 was detectedby Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels werealso detected and served as a loading control.

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Fig. 5.Anti-tumor activity of cucurbitacin I and its PEO-b-PBCL micellar formulation in B16melanoma tumor-bearing mice. Mice bearing B16 melanoma tumor at their left flank receivedintratumoral injection of 1 mg/kg/day cucurbitacin I, solubilized in 20% ethanolic solution orencapsulated in polymeric micelles for 10 days. (A) average tumor volume in animals duringtreatment; (B) average tumor weight in each group at the end point of the experiment (day 15and 17 after tumor implantation in control and test groups, respectively); and (C) photographsof tumor-bearing animals at the end of experiment: (C1) untreated, (C2) treated with emptymicelles, (C3) soluble cucurbitacin I, and (C4) polymeric micellar cucurbitacin I.

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Molavi et al. Ta

ble

1C

hara

cter

istic

s of c

ucur

bita

cin-

load

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= 3)

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5000

–240

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065

± 0.

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65.1

± 3

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26 ±

0.0

1

PEO

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BC

L 50

00–4

700

0.09

2 ±

0.00

592

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4.6

76.3

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0.26

± 0

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68.4

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17 ±

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4 ±

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Molavi et al.

Table 2The IC50 values of free and polymeric micellar cucurbitacins against B16.F10 cells (n = 3)

Drug Vehicle IC50 ± S.D. (μM)

24 h 48 h

Cucurbitacin B 2% methanol 18.9 ± 2.1 16.1 ± 2.1

5000–24,000 52.9 ± 2.8a 17.1 ± 0.3

PEO-b-PCL micelles

Cucurbitacin I 2% methanol 4.7 ± 1.6b 4.6 ± 0.1b

5000–24,000 9.7 ± 1.1a,b 5.9 ± 0.5a,b

PEO-b-PCL micelles

aSignificantly different from free drug (P< 0.05).

bSignificantly different from cucurbitacin B (P< 0.05).


polymeric micelles for the solubilization and delivery of stat3 inhibitor cucurbitacins in solid...

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